1. Field of the Invention
The present invention relates to methods and apparatus for delivering molecules into a target cell, and, more particularly, to such methods and apparatus for achieving such delivery through electroporation and electromigration.
2. Description of Related Art
The effect of electromagnetic fields on cell membranes has been studied since the 1960s. Early research focused on describing observations that an applied electric field can reversibly break down cell membranes in vitro. Throughout the 1970s the topic was more common in the literature and continued to focus on describing the phenomenon that resulted from brief exposure to intense electric fields as well as the entry of exogenous molecules to the cell interior as a result of membrane breakdown. Applications began to emerge along with a better understanding of reversible membrane breakdown in the 1980s.
Prior research led to the current understanding that exposure of cells to intense electric fields for brief periods of time temporarily destabilized membranes. This effect has been described as a dielectric breakdown due to an induced transmembrane potential, and was termed xe2x80x9celectroporation,xe2x80x9d or xe2x80x9celectropermeabilization,xe2x80x9d because it was observed that molecules that do not normally pass through the membrane gain intracellular access after the cells were treated with electric fields. The porated state was noted to be temporary. Typically, cells remain in a destabilized state on the order of minutes after electrical treatment ceases.
The physical nature of electroporation makes it universally applicable. A variety of procedures utilize this type of treatment, which gives temporary access to the cytosol. These include production on monoclonal antibodies, cell-cell fusion, cell-tissue fusion, insertion of membrane proteins, and genetic transformation. In addition, dyes and fluorescent molecules have been used to investigate the phenomenon of electroporation. A notable example of loading molecules into cells in vivo is electrochemotherapy. The procedure utilizes a drug combined with electric pulses as a means for loading tumor cells with an anticancer drug, and has been performed in a number of animal models and in clinical trials by the present inventors. Also, plasmid DNA has been loaded into rat liver cells in vivo (Heller et al., FEBS Lett. 389, 225-28).
Protocols for the use of electroporation to load cells in vitro typically use a suspension of single cells or cells that are attached in a planar manner to a growth surface. In vivo electroporation is more complex because tissues are involved. Tissues are composed of individual cells that collectively make up a three-dimensional structure. In either case, the effects on the cell are the same. FIG. 1 illustrates details of the electrical treatment procedure. Electrodes and electrode arrays for delivering electrical waveforms for therapeutic benefit, including inducing electroporation, have been described by Bernard (WO 98/47562).
The loading of molecules by electroporation in vitro as well as in vivo is typically carried out by first exposing the cells or tissue of interest to a drug or other molecule (FIG. 2). The cells or tissue are then exposed to electric fields by administering one or more direct current pulses. Electrical treatment is conducted in a manner that results in a temporary membrane destabilization with minimal cytotoxicity. The intensity of electrical treatment is typically described by the magnitude of the applied electric field. This field is defined as the voltage applied to the electrodes divided by the distance between the electrodes. Electric field strengths ranging from 1000 to 5000 V/cm have been used for delivering molecules in vivo and are also specific to the cells or tissue under investigation. Pulses are usually rectangular in shape; however, exponentially decaying pulses have also been used. The duration of each pulse is called pulse width. Molecule loading has been performed with pulse widths ranging from microseconds (xcexcs) to milliseconds (ms). The number of pulses delivered has ranged from one to eight. Typically, multiple pulses are utilized during electrical treatment.
For molecules to be delivered to the cell interior by electroporation, it is important that the molecule of interest be near the exterior of the cell membrane when in the cell is in a permeabilized state. It is also important to have molecules near substantially all cells within a treated tissue volume in order to provide efficient delivery to substantially all cells within the treatment volume.
Currently, molecules are injected systemically, via methods well known to those of skill in the art, or directly into the treatment site. No attempt is made to produce a specific distribution.
These methods do not ensure that the distribution of molecules is sufficient to provide effective delivery to substantially all the cells.
Electropermeabilization of tumor cell membranes in vivo has been reported (Rols et al., Nature Biotechnology 16, 173, 1998) using applied electric pulses from surface electrodes in contact with the skin. A protein can be transferred into or and expressed by the cells by incorporating either the protein or a plasmid carrying a reporter gene. The efficiencies of transfer for the protein and plasmid were, respectively, 20 and 4%.
A first type of electrode known in the art comprises parallel-plate electrodes placed on opposite sides of the tumor. Other electrodes known in the art at the present time comprise needles that are inserted into or around the tissue of interest. Electric fields are applied in only two dimensions of the three-dimensional tissue matrix. This limits the area of each cell that can be electroporated (FIG. 1), which reduces delivery efficiency.
A two-dimensional array of needles has also been disclosed (Gilbert et al., Biochim. Biophys. Acta 1334, 9, 1997; U.S. Pat. No. 5,702,359) in which circularly disposed pairs of needles surround a target tissue. Pulses of opposite polarity are applied across each pair of needles in a predetermined sequence, which has been shown to improve tumor regression in a mouse melanoma study.
Electrodes and methods known in the art do not provide molecule movement during the preelectroporation period to enhance molecular distribution nor in the postelectroporation time period, when the cells are in a state of increased membrane permeability. The movement of molecules within the tissue is believed to effect an increase in the delivered quantity of molecules by enhancing movement into the cells.
It is therefore an object of the present invention to provide an improved device and method for manipulating molecules adjacent and/or within a target tissue site.
It is an additional object to provide such a device and method for manipulating molecules in three-dimensional space such as a tissue volume element.
It is a further object to provide such a device and method that can provide a desired electromagnetic field distribution adjacent and/or within a target tissue.
It is another object to provide such a device and method that can be configured to activate a multicomponent labile system at a desired site.
It is yet an additional object to provide a system for effecting tumor reduction.
It is yet a further object to provide a system for effecting in vivo gene transfer to cells via electroporation.
These objects and others are attained by the present invention, a device for manipulating a molecule in vivo relative to a target tissue in three dimensions. The device comprises a support and at least one member affixed to and extending away from the support. The member has at least two discrete electrodes, each electrode in independent circuit communication with a respective portion of a source of electrical energy and therefore being differentially activatable.
The discrete electrodes are configured to establish a first electromagnetic field in vivo between selected electrodes sufficient to manipulate a molecule translationally relative to a target tissue. The electrodes are farther configured to establish a second electromagnetic field sufficient to cause transient permeability of a cell membrane within the target tissue. In a particular embodiment the electrodes are also configured to establish a third electromagnetic field sufficient to manipulate a molecule translationally following the second field to continue enhancing molecular distribution and/or uptake at cells. Typically the first and the third field levels will be lower than that of the second, although this is not intended as a limitation.
In an alternate embodiment, a system includes at least two members extending from a support. A first member includes at least one electrode as described above; a second member includes at least two electrodes. Such an arrangement permits triangulation between the independently activatable electrodes.
In a particular embodiment the electrodes are activatable in a predetermined sequence, which may include sequential or simultaneous activation of any or all of the electrodes.
The device can be used, for example, with alternating current, direct current, pulsed alternating current, pulsed direct current, high- and low-voltage alternating current with variable frequency and amplitude, variable direct current waveforms, variable alternating current signals biased with variable direct current waveforms, and variable alternating current signals biased with constant direct current.
Several embodiments of the methods of the present invention include the use of a device as described above to enhance the delivery of a molecule such as a bioactive molecule, nucleic acid, amino acid, polypeptide, protein, antibody, glycoprotein, enzyme, oligonucleotide, plasmid DNA, chromosome, or drug, although this list is not intended to be exhaustive or limiting. In a related embodiment the device may be used to cause the electromigration of at least two components of a multicomponent reactive system into apposition to permit a reaction to occur at a desired target tissue site. The target tissue may comprise a tumor, and organ, or wound site.
The features that characterize the invention, both as to organization and method of operation, together with further objects and advantages thereof, will be better understood from the following description used in conjunction with the accompanying drawing. It is to be expressly understood that the drawing is for the purpose of illustration and description and is not intended as a definition of the limits of the invention. These and other objects attained, and advantages offered, by the present invention will become more fully apparent as the description that now follows is read in conjunction with the accompanying drawing.